Summary. The kinetics of adherence of single isolates of Staphylococcusaureus, S. epidermidis, Pseudomonas aeruginosa and Escherichia coli to catheters made of polyvinyl chloride (PVC), Teflon@,siliconised latex, polyurethane and Vialon@was evaluated by a radiometric assay. Radiolabelled bacteria (lo8 cfulml) were incubated in vials containing 1-cm lengths of catheter for up to 3 days. The peak of maximal adherence to each biomaterial was reached after 24 h for P . aeruginosa and after 72 h for the other strains. Bacterial adherence to PVC and siliconised latex was significantly higher (2-6 times; p<O.O5) than to the other biomaterials for all the strains. The lowest values of adherence were observed with polyurethane and Vialon@ the staphylococci but with Teflon@ E. coli and P . aeruginosa. for for Bacterial viability and growth was evaluated in eluates obtained from incubation of segments of each catheter in buffer for 24 h. None of the eluates affected the viability of the staphylococci. However, all of them, significantly increased the growth of E. coli and P . aeruginosa with the exception of the eluate from siliconised latex, in which the inoculum count was reduced to an undetectable level for E. coli. We conclude that bacterial adherence to catheters may depend in part on the nature of the biomaterial and that certain substances eluted from the catheters may affect the viability and growth of different micro-organisms.

Introduction
Intravascular and urinary catheters are frequently used in hospitals. Several studies have demonstrated significant infection rates associated with local skin conditions, methods used in the placement of such devicesand the duration of catheter usage. Catheterassociated infections are a significant source of nosocomial morbidity and m~rtality.~ mechaThe nisms by which infections develop are not fully understood but the phenomenon of adherence to the biomaterials appears to be a critical factor in initiating colonisation and subsequent infe~tion.~ The nature and chemical composition of the biomaterials commonly used to make medical devices differ, and many of them contain different additives and plasticisers to improve their physico-chemical properties and biocompatibility.’ The adherence of different micro-organisms to, and their survival in, catheters is promoted not only by bacterial factors but additional bacterium-device interactions could participate in this phenomenon.6 It has been proposed that some micro-organisms, e.g., coagulase-negative staphylococci, could metabolise some of the components of plastic catheters in the absence of other nutrients and use them to sustain growth on the surface of biomaterials.’
Received 3 May 1990; revised version accepted 9 Oct. 1990. *Correspondenceshould be sent to Dr A. Pascual.
349

We have developed an experimental in-vitro system for evaluating quantitatively the adherence of bacteria over a period of 3 days to five intravascular and urinary catheters composed of polyvinyl chloride (PVC), Teflon@, siliconised latex, polyurethane and Vialon@. The effect of eluates of these biomaterials on the growth of different micro-organisms in the absence of other nutrient sources has also been assessed.

Materials and methods
Bacterial strains and radioactive labelling
One strain each of Escherichia coli (HUS 25), Pseudomonas aeruginosa (HUS 36), Staphylococcus aureus (HUS 41) and S . epidermidis (HUS 59) were used. The strains were isolated from blood cultures of patients with catheter-associated infections. Strains were identified by standard methods. Heavy suspensions of each strain in Tryptone Soy Broth (Oxoid) with glycerol 10% were stored in small volumes at 70°C. For adherence assays, several colonies from a nutrient-agar plate were inoculated into 5 ml of Mueller-Hinton broth containing (2-3H) adenine (specific activity 24 Ci/mmol; Amersham) 10 ~ 1 . ~ Before use, bacteria were washed three times with phosphate-buffered saline (PBS) and resuspended to obtain a final concentration of lo8 cfu/ml.

. coli to catheters of different material i. Vialon@. For adherence assays. coli and staphylococci (table I). 6.m. U. 1. aureus and P . Eire) . After incubation for 24 h. 0 . catheters were cut under sterile conditions into 1. The peak of bacterial adherence was reached between 24 and 72 h for E.05. The lowest adherence values were
. The Netherlands). and (e) Vialon@(Viacath@.
adherence of both micro-organisms was significantly higher to PVC and siliconised latex than to Teflon@. bar=SD): PVC. aeruginosa and staphylococci (table I).
Bacterial growth in catheter eluates
Each strain was inoculated into catheter eluates at a final concentration of lo3 cfu/ml. siliconised latex. aeruginosa adhered to the biomaterial more rapidly. 10-pl samples were diluted in ice-cold PBS and pour plates made in Mueller-Hinton Agar (Oxoid) . J. (Biovial@. (c) latex (siliconised) two-way paediatric Foley catheter (Inmed@.' Eluates of the catheters were prepared by incubating 30 segments of each in 10 ml of PBS at 37°C for 24 h under sterile conditions.colonies were counted after incubation for 48 h at 37°C. two catheter segments were removed. USA). coli to the plastic catheters up to 72 h of incubation are shown in figs. 1 and 2. Lumac/3M. Malaysia). USA) with 2. (n=3. H.350
G.
Statistical methods
Results were expressed as mean and SD. . A.Teflon". Braun Melsugen AG. 8. After 5 min. coli to both biomaterials was similar but was significantly greater than that observed with Teflon@. siliconised latex. However.O-cm segments and pre-incubated in lOml of sterile isotonic PBS at 37°C for 1 h. Becton Dickinson. Inc). B. bar=SD). The adherence of E. (b) Teflon@ (Abbocath R-T. At timed intervals. epidermidis to catheters of different i.H. PEREA
Cathe ters
Catheters made of five different biomaterials were used : (a) polyvinyl chloride (PVC) (Drum-Cartridge@ catheter. Germany). N. polyurethane or Vialon@.polyurethane.5ml of scintillation fluid (Aqua Luma Plus. P . PASCUAL AND E. Maximal bacterial adherence was observed with PVC followed by siliconised latex for P .
Results
Bacterial adherence to catheters
The kinetics of adherence of S. the number of bacteria adherent per cm2 of catheter was calculated by dividing the number of bacteria adherent to a catheter piece by the total surface area of the piece. 1Oml of radioactive bacterial suspension (10' cfulml) were incubated with 30 segments of each catheter type in 25-ml screwcapped glass vials at 37°C. polyurethane and Vialon@. (d) polyurethane (Cavafix Certo@. Abbott Laboratories) . Catheter-bound radioactivity was determined in a scintillation counter (1701 LS Beckman Instrument. materials (n=3. Differences amongstgroups were compared by analysisof variance and the Bonferroni method" was used to assess statistical significance at p < 0. Vialon".Inmed USA. epidermidis and E. aeruginosa. Teflon". . LOPEZ-LOPEZ. Beckman Instruments. polyurethane. 24 and 72 h. The relationship between radioactivity and number of bacteria was obtained by counting in a biovial 100 pl of the initial inoculum. reaching maximal adherence between 6 h for PVC and 24 h for the other biomaterials.
F g 1 Mean adherence of S.
F g 2 Mean adherence of E. washed five times in cold PBS to remove all non-adherent bacteria and deposited in polypropylene vials.
.. From this value.
Bacterial adherence to urinary catheters
For the adherence assays. Abbott Laboratories. PVC. Similar behaviour was observed with S. 0 .

9 (0.PLASTIC MATERIALS A N D BACTERIA
351
Table I. ) 06t 5.
Bacterial growth in catheter eluates
The growth rates of bacteria in eluates obtained from the incubation of catheter segments in PBS for 24 h at 37°C are shown in tables I1 and 111. aeruginosa showed that. at 24 h. but marked differences developed after 24 h. depending on the strain and the catheter material involved. aeruginosa
26. Observations (unpublished) with different strains of P . Maximum levels of bacterial adherence to five types of plastic catheter during incubation for 72 h at 37°C
Catheter material Siliconised latex PVC Teflon@ Polyurethane Vialon@
I
I
Mean* (SD) number of adherent bacteria (105/cm2)
S .9( .12 chemiluminescence' or the use of radiolabelled
bacteria.2) 15.
Discussion
Bacterial adherence to prosthetic materials can be measured by quantitative culture. t p < 0.0)t 40. aeruginosa (table 11) after incubation for 24 h.8(3*8)$ 9. *Mean of three experiments. adherence was measured over a longer period of time than is usual in such studies.5 (0.1) 6.0(0-4)t 0. which are mainly mediated by attracting and repelling forces such as hydrophobic interactions between the two surfaces.8) 18. Growth of E. coli and P. aeruginosa
24 h 15 (0.2)
5.9(2.
obtained with Teflon@for gram-negative bacteria. However. The increased adherence measured may have been due to microcolony formation and growth may be required for this adherence to occur.7) 22. Teflon@. Further studies are needed with scanning electronmicroscopy to compare the quantitative data with morphological studies. epidermidis and Vialon@for S.6 The latter method has provided a simple and reproducible method for quantifying the bacterial biomass on the surface of biomaterials. However.2) 7. epidermidis after incubation for 24 h.8 (0-1) 0 1. aureus or S .1 (0. in siliconised latex eluates the number of viable organisms was reduced to undetectable levels (< 10 cfu/ ml). epidermidis. $after 6 h.8)t 60.1) 9.1 ( . Very little difference in adherence was shown at 1 h or 6 h. whereas adherence in this study was similar for both types of catheter material.5 (1-2)t 11-5( .9(1.6 (3. ) 10t 13. All the eluates stimulated the growth of P .4) 3.
.8( . After incubation for 24 h.1 (1. numbers of E.0(2. initial inoculum lo3 cfu/ml.8) 6.6 This method is particularly useful for measuring the initial interactions between bacteria and polymers.0 (4.5 (1.4) 42.6 In our study.3(0. bacteria on a PVC catheter are immersed in an amorphous material which is not observed with polyurethane.6 (0-2)
7-5 (1.0(10. Different results were observed with staphylococci (table 111).6) 11. 0 1 1 -0( .
aureus. only the Teflon@ eluate significantly decreased the viable number of S.8) 2.3)t 250. coli were significantly higher in PVC.0 (19.4) 7.8(0. ) 03t
18. Our results indicate that catheter material type may be important in adherence of bacteria to catheter
Table 11.9 (2.8 (2.5 (1.6) .5) 4. epidermidis
E. ) 12t
*Mean of three experiments. coli
P . lack of correlation between longer term adherence and growth in catheter eluates suggests that factors other than growth due to soluble catheter components is involved.5 (0.5(1-7) 7.aeruginosa in eluates from different catheters
Mean (SD) number of cfu (104/ml) Catheter material 6h None (control) Siliconised latex PVC Teflon@ Polyurethane Vialon@
E.2) 1-5 (0-2)t 2.4(3.6 (1. aureus
S . polyurethane for S. polyurethane and Vialon@eluates than in the controls.9)
17.4(1.0(58)t
6h
24 h 600 (31-1) 1150(lll)t 3150 (320)t 2980 (262)t 3250 (342)t 3506 (298)t
0.5)
0= c 10 cfulml.' microscopy.05 compared to the controls. None of the catheter eluates significantly increased the growth of either S.8) 6. ) 22t 160.2)
24. coli P . ?Maximum adherence measured after incubation for 24 h.

5 (0. 70: 719-732.1) 0. Teflon@and both offered the greatest resistance to adherence of staphyl. ingestion and killing by human polymorphonuclearleukocytes: a quantitative assay using
5 . coli but increased the growth of which measures viable bacteria.we tested the effectof catheter This study was partly supported by grant no. coli and P . i 2. McKeel DW. aureus or S . siliconised latex. J Infect Dis 1982. PA 85-0299 from the Direccibn General de Investigacibn Cientifica y Tkcnica components on bacterial growth. 146:479-482. Welch GW.2) 0-3 (0-1) 0.4) 0.0 (0. PVC. Am JMed 1981.in the manufacturing other surface properties. None of the eluates tested increased the initial adherence of coagulase-negative staphylococci growth of either S . Some of these substances vivo because the catheters are rapidly coated with could be eluted from the catheter into the medium and different proteins. Rhame FS. J Infect D s 1980.3) 0-5 (0. Corrado ML. 3.4 (0-2)
1. Infection control in intravenous therapy. PEREA
Table 1 1 Growth of S . A. Surg Gyn Obstet 1974.2(0.7 (0. contain species because it is known that there is variation several additives to make them flexible enough to be between strains of a species both in adherence and in used in catheters. attachment.2 (0-09) 0.6 (0. Peterson PK.tigation to define the mechanisms involved in the ococci adherent to intravascular catheters in the pathogenesis of catheter-related infections. Pulverer G. J. Nosocomial bacteriemia: an epidemiologic overview. eluate was toxic for E. Locci R.aureus
6h 0. 141: 781-787.1) 0. The results obtained with seems less likely that they would grow in a catheter single isolates cannot be extrapolated to the whole eluate in PBS. Growth of E. Franson TR. C. aureus in eluates from different catheter 1. J Clin Microbioll986.polyurethanes was significantly greater than that ococci and Teflon@for E.3) 0. Quantitative culture of intravenous catheters and other intravascular inserts. fluids and cells.3) 0. Quie PG. Westerdaal NAC. The role
. Verhoef J. 4. siliconised latex Previous studies' ' with a blood-agar-roll technique.g.2 (0. Seligman SJ. Persistent in vitro survivalof coagulase-negativestaphylococciadherent to intravascular catheters in the absence of conventional nutrients. 24: 559-561. Although these findings require further characteradherence may depend mainly on the catheter bioisation by more extensive in-vitro and clinical invesmaterial. aeruginosa. Silverstein P.
References
1. Staphyloto PVC catheters was also greater than that observed cocci have complex nutritional requirements and it with Teflon@catheters. However. We thank M.2) 0. have shown that the P . several chemicals are used and the exact The measurement of bacterial adherence to clean catheters is a simplification of the events that occur in formulae are unknown.3) 0. 6. Peters G. e.2) 24 h 0. LOPEZ-LOPEZ. Maki DG. Walker HL. organisms to become metabolically dormant. Many biomaterials.2 (0. process. Cleri DJ. adherence to the surface of foreign objects may allow colonisation.2 (0. Sohnle PG. Guzman for her technical obtained from the incubation of catheter segments in assistance and Sandra Hidalgo for assistance with the preparation PBS as growth media. Menon L. epidermidis.4 (0. Goldman DA.7 (0. The survival in vitro of coagulase-negative staphyl. Ann Intern Med 1973.3) 0. 8. Pascual A.08) 0.5 : 5 18-522.2) 0. 7.g.2 (0. Sheth NK.04) 0. of the manuscript. Others. Modulation of adherenceof coagulase-negativestaphylococcito Teflon catheters in vitro. Maki DG..
of catheter composition in the development of thrombophlebitis. materials
Mean (SD) number of cfu (103/ml)
I
Catheter material 6h None (control) Siliconised latex PVC Teflon@ Polyurethane Vialon@ See footnotes to table 11.3) 1..
S. observed in the controls. come from different tropical countries. Kinetics of staphylococci opsonization.79: 867-887. 138:421-424. We used eluates (DGICYT. Adherence and growth of coagulase-negative staphylococci on surfaces of intravenous catheters. Verhoef J.7 (0-3) 0. epidermidis
S. but the initial could be used by different micro-organisms. preliminary data suggest that the effect of new It has been proposed that these micro-organismscould biomaterials on bacterial adherence and viability use some of the components of the catheters as should be evaluated before clinical trials to obtain nutrients for g r ~ w t h It~ may also be possible that catheters that could prevent bacterial adherence and . polyurethane and Vialon@ aeruginosa in eluates from PVC. Spain).1 (0.09)
24 h 0-6(0. coli and P . aeruginosa. To consider this hypothesis.6 (0-2) 0. Fleer A.06)t 0. e. PASCUAL AND E. epidermidis and S .08) 0. these absence of conventional nutrients has been described.5 (0. Eur J Clin Microbioll986.0 (0. In our assays.6 (0-2)
surfaces.352
G.1 (0.6 (0.